Signal Peptide for Producing a Polypeptide

ABSTRACT

The present invention relates to a method for producing a polypeptide comprising using a signal peptide, to nucleic acid constructs comprising a first nucleotide sequence encoding the signal peptide and a second nucleotide sequence encoding a polypeptide which is foreign to the first nucleotide sequence. Furthermore, it also relates to expression vectors and host cells comprising the nuclei acid construct.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/357,223filed Jan. 24, 2012, which is a continuation of U.S. application Ser.No. 12/415,174 filed Mar. 31, 2009 (now U.S. Pat. No. 8,124,379), whichis a continuation of U.S. application Ser. No. 11/152,811 filed Jun. 14,2005 (now U.S. Pat. No. 7,527,947), which claims priority or the benefitunder 35 U.S.C. 119 of Danish Application no. PA 2004 00917 filed Jun.14, 2004 and U.S. provisional application No. 60/580,151 filed Jun. 16,2004, the contents of which are fully incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

The present application contains information in the form of a sequencelisting, which is appended to the application and also submitted on adata carrier accompanying this application. The content of the datacarrier is fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to methods for producing a polypeptidecomprising using a signal peptide foreign to the polypeptide, nucleicacid constructs comprising a first and a second nucleotide sequenceencoding the signal peptide and the polypeptide and expression vectorsand host cells comprising said nucleic acid construct.

BACKGROUND OF THE INVENTION

The recombinant production of a heterologous protein in a fungal hostcell, particularly a filamentous fungal cell such as Aspergillus or ayeast cell such Saccharomyces, may provide for a more desirable vehiclefor producing the protein in commercially relevant quantities.

Recombinant production of a heterologous protein is generallyaccomplished by constructing an expression cassette in which the DNAcoding for the protein is placed under the expression control of apromoter, excised from a regulated gene, suitable for the host cell. Theexpression cassette is introduced into the host cell. Production of theheterologous protein is then achieved by culturing the transformed hostcell under inducing conditions necessary for the proper functioning ofthe promoter contained on the expression cassette.

Improvement of the recombinant production of proteins generally requiresthe availability of new regulatory sequences which are suitable forcontrolling the expression of the proteins in a host cell.

It is an object of the present invention to provide improved methods forproducing a polypeptide in a fungal host cell using signal peptidesequences.

SUMMARY OF THE INVENTION

The invention provides a method for producing a secreted polypeptide,comprising:

(a) cultivating an fungal host cell in a medium conducive for theproduction of the polypeptide, wherein the host cell comprises a nucleicacid construct comprising a first nucleotide sequence encoding a signalpeptide operably linked to a second nucleotide sequence encoding thepolypeptide, wherein the first nucleotide sequence is foreign to thesecond nucleotide sequence, the 3′ end of the first nucleotide sequenceis immediately upstream of the second nucleotide sequence, and the firstnucleotide sequence is selected from the group consisting of:

-   -   (i) a nucleotide sequence encoding a signal peptide having an        amino acid sequence which has at least 70% identity with SEQ ID        NO:1;    -   (ii) a nucleotide sequence having at least 70% homology with SEQ        ID NO: 2; and    -   (iii) a nucleotide sequence which hybridizes under stringency        conditions with the nucleotides of SEQ ID NO: 2, or its        complementary strand, wherein the stringency conditions are        defined as prehybridization, hybridization, and washing        post-hybridization at 5° C. to 10° C. below the calculated Tm in        0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40,        1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium        monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml,        and washing once in 6×SCC plus 0.1% SDS for 15 minutes and twice        each for 15 minutes using 6×SSC at 5° C. to 10° C. below the        calculated Tm; and

(b) isolating the secreted polypeptide from the cultivation medium.

Furthermore, the present invention provides for A nucleic acid constructcomprising a first nucleotide sequence encoding a signal peptideoperably linked to a second nucleotide sequence encoding a polypeptide,wherein the first nucleotide sequence is foreign to the secondnucleotide sequence, and the 3′ end of the first nucleotide sequence isimmediately upstream of the second nucleotide sequence, and the firstnucleotide sequence is selected from the group consisting of:

(a) a nucleotide sequence encoding a signal peptide having an amino acidsequence which has at least 70% identity with SEQ ID NO:1;

(b) a nucleotide sequence having at least 70% homology with SEQ ID NO:2; and

(c) a nucleotide sequence which hybridizes under stringency conditionswith the nucleotides of SEQ ID NO: 2, or its complementary strand,wherein the stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at 5° C. to 10° C. belowthe calculated Tm in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5%NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodiummonobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml, andwashing once in 6×SCC plus 0.1% SDS for 15 minutes and twice each for 15minutes using 6×SSC at 5° C. to 10° C. below the calculated Tm.

The present invention also provides expression vectors and host cellscomprising said nucleic acid construct.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “variant” when used in reference to another polypeptide ornucleotide sequence is in the context of the present invention to beunderstood as a polypeptide or nucleotide sequence which comprises asubstitution, deletion, and/or insertion of one or more amino acids ornucleotides as compared to another polypeptide (i.e. it is a variant ofpolypeptide/nucleotide sequence it is compared with). In particular thechanges may be of minor nature, such as conservative amino acidsubstitutions or for nucleotide sequence resulting in conservative aminoacid substitutions, that do not significantly affect the activity of thepolypeptide; or small deletions, typically of one to about 20 aminoacids depending on the size of the polypeptide in which the changes aremade.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter the specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly as well as these inreverse.

Method of the Present Invention

The present invention relates to methods for producing a secretedpolypeptide, comprising: (a) cultivating a fungal host cell in a mediumconducive for the production of the polypeptide, wherein the host cellcomprises a nucleic acid construct comprising a first nucleotidesequence encoding a signal peptide operably linked to a secondnucleotide sequence encoding the polypeptide, wherein the firstnucleotide sequence is foreign to the second nucleotide sequence, the 3′end of the first nucleotide sequence is immediately upstream of thesecond nucleotide sequence, and the first nucleotide sequence isselected from the group consisting of: (i) a nucleotide sequenceencoding a signal peptide having an amino acid sequence which has atleast 70% identity with SEQ ID NO:1; (ii) a nucleotide sequence havingat least 70% homology with SEQ ID NO: 2; and (iii) a nucleotide sequencewhich hybridizes under stringency conditions with the nucleotides of SEQID NO: 2, or its complementary strand, wherein the stringency conditionsare defined as prehybridization, hybridization, and washingpost-hybridization at 5° C. to 10° C. below the calculated Tm in 0.9 MNaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1×Denhardt'ssolution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate,0.1 mM ATP, and 0.2 mg of yeast RNA per ml, and washing once in 6×SCCplus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSCat 5° C. to 10° C. below the calculated Tm; and (b) isolating thesecreted polypeptide from the cultivation medium.

In the methods of the present invention, the fungal host cells arecultivated in a medium conducive for the production of the polypeptide,i.e. in a nutrient medium suitable for production of the polypeptideusing methods known in the art. For example, the cell may be cultivatedby shake flask cultivation, or small-scale or large-scale fermentation(including continuous, batch, fed-batch, or solid state fermentations)in laboratory or industrial fermentors performed in a suitable mediumand under conditions allowing the polypeptide to be expressed and/orisolated. The cultivation may take place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection).

The polypeptide may be detected using methods known in the art that arespecific for the polypeptide. Such detection methods may include use ofspecific antibodies, formation of an enzyme product, or disappearance ofan enzyme substrate.

In the methods of the present invention, the fungal host cell may inparticular produce at least about 25% more, more particularly at leastabout 50% more, more particularly at least about 75% more, moreparticularly at least about 100% more, even more particularly at leastabout 200% more, most particularly at least about 300% more, and evenmost particularly at least about 400% more polypeptide relative to afungal cell containing a native signal peptide sequence operably linkedto a nucleotide sequence encoding the polypeptide when cultured underidentical production conditions.

The resulting secreted polypeptide can be recovered directly from themedium by methods known in the art. For example, the polypeptide may berecovered from the nutrient medium by conventional procedures including,but not limited to, centrifugation, filtration, extraction,spray-drying, evaporation, or precipitation.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989).

Signal Peptide

The first nucleotide sequence of the present encodes a signal peptide ofthe present invention. The term “signal peptide” or “signal peptidesequence” is defined herein as a peptide sequence usually present at theN-terminal end of newly synthesized secretory or membrane polypeptideswhich directs the polypeptide across or into a cell membrane of the cell(the plasma membrane in prokaryotes and the endoplasmic reticulummembrane in eukaryotes). It is usually subsequently removed. Inparticular said signal peptide may be capable of directing thepolypeptide into a cell's secretory pathway.

The term “operably linked” is defined herein as a configuration in whicha control sequence, e.g., a signal peptide sequence, is appropriatelyplaced at a position relative to a coding sequence such that the controlsequence directs the production of a polypeptide encoded by the codingsequence.

The term “coding sequence” is defined herein as a nucleotide sequencewhich is translated into a polypeptide when placed under the control ofthe appropriate control sequences. The boundaries of the coding sequenceare generally determined by the start codon located at the beginning (5′end) of the open reading frame and a stop codon located at the 3′ end ofthe open reading frame. A coding sequence can include, but is notlimited to, genomic DNA, cDNA, RNA, semisynthetic, synthetic, andrecombinant nucleotide sequences.

The 5′ end of the coding sequence of the polypeptide of the presentinvention may contain a native nucleotide sequence encoding a signalpeptide which is naturally linked with nucleotide sequence segment whichencodes the mature (or pro-form) of the polypeptide. In this case thesignal peptide of the present invention may replace the native signalpeptide. Alternatively, the polypeptide of the present invention maylack a native signal peptide. In this context the term “native” isintended to be understood as being present naturally.

In the methods of the present invention, the signal peptide sequence isforeign to the nucleotide sequence encoding a polypeptide of interest,but the signal peptide sequence or nucleotide sequence may be native tothe fungal host cell. In this context the term “foreign” is intended tobe understood as the signal peptide is not native to the polypeptide,i.e. it originates from another gene than the polypeptide.

In one embodiment the first nucleotide sequence may encode a signalpeptide having an amino acid sequence which has at least 70%,particularly at least about 75%, more particularly at least about 80%,more particularly at least about 85%, even more particularly at leastabout 90%, most particularly at least about 95%, and even mostparticularly at least about 97% identity to SEQ ID NO: 1, which have theability to direct a polypeptide into or across a cell membrane(hereinafter “homologous signal peptide”), e.g. into a cell's secretorypathway. In a particular aspect, the homologous signal peptide may havean amino acid sequence which differs by five amino acids, particularlyby four amino acids, more particularly by three amino acids, even moreparticularly by two amino acids, and most particularly by one amino acidfrom SEQ ID NO: 1. For purposes of the present invention, the identityor degree of identity between two amino acid sequences is determined bythe Clustal method (Higgins, 1989, CABIOS 5: 151-153) using theLASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.) with anidentity table and the following multiple alignment parameters: Gappenalty of 10 and gap length penalty of 10. Pair wise alignmentparameters are Ktuple=1, gap penalty=3, windows=5, and diagonals=5.

In particular, the first nucleotide sequence may encode a signal peptidewhich comprises the amino acid sequence of SEQ ID NO: 1, or an allelicvariant thereof; or a fragment thereof that has the ability to directthe polypeptide into or across a cell membrane, e.g. into a cell'ssecretory pathway. In a more particular aspect, the first nucleotidesequence of the present invention encodes a signal peptide thatcomprises the amino acid sequence of SEQ ID NO: 1. In another particularaspect, the first nucleotide sequence encodes a signal peptide thatconsists of the amino acid sequence of SEQ ID NO: 1, or a fragmentthereof, wherein the signal peptide fragment has the ability to direct apolypeptide into or across a cell membrane, e.g. into a cell's secretorypathway. In another more particular aspect, the nucleotide sequence ofthe present invention encodes a signal peptide that consists of theamino acid sequence of SEQ ID NO: 1.

The present invention also encompasses first nucleotide sequences whichencode a signal peptide having the amino acid sequence of SEQ ID NO: 1,which differs from SEQ ID NO: 2 by virtue of the degeneracy of thegenetic code. The present invention also relates to subsequences orfragments of SEQ ID NO: 2 which encode fragments of SEQ ID NO: 1 whichhas the ability to direct a polypeptide into or across a cell membrane,e.g. into a cell's secretory pathway.

A subsequence of SEQ ID NO: 2 is a nucleic acid sequence encompassed bySEQ ID NO: 2 except that one or more nucleotides from the 5′ and/or 3′end have been deleted. Particularly, a subsequence contains at least 30nucleotides, such as at least 35 nucleotides or at least 40 nucleotidesor at least 45 nucleotides or at least 50 nucleotides or at least 52nucleotides or at least 53 nucleotides. A fragment of SEQ ID NO: 1 is apolypeptide having one or more amino acids deleted from the amino and/orcarboxy terminus of this amino acid sequence. In particular a fragmentcontains at least 10 amino acid residues. such as at least 12 amino acidresidues or at least 13 amino acid residues or at least 14 amino acidresidues or at least 15 amino acid residues or at least 16 aminoresidues or at least 17 amino acid residues. In particular if the firstand second nucleotide sequences are expressed in a yeast host cell thesignal peptide may comprise amino acid residues 1-16 of SEQ ID NO: 1, asthe inventors of the present invention have observed that in yeast thesignal peptide is cleaved between amino acid residues 16 and 17 (at theAA↓LP), while if the host cell is a filamentous fungus the signalpeptide may in particular comprise the 18 amino acid residues of SEQ IDNO: 1, as the inventors of the present invention have seen that inAspergillus oryzae the cleavage of the signal peptide occurs after aminoacid residue 18 of SEQ ID NO:1. This indicates that there may be adifference between yeast and filamentous fungi in the sequencerecognized as a cleavage site for cleavage of the signal sequence.

An allelic variant denotes any of two or more alternative forms of agene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations may be silent (no change in the encodedsignal peptide) or may encode signal peptides having altered amino acidsequences. The allelic variant of a signal peptide is a peptide encodedby an allelic variant of a gene.

In a particular aspect, the first nucleotide sequence is the signalpeptide coding sequence of the cutinase gene contained in Humicolainsolens DSM 1800 (SEQ ID NO: 1).

In a second aspect, the first nucleotide sequence of the presentinvention which encodes a signal peptide may have a degree of homologyto SEQ ID NO: 2 of at least about 70%, particularly at least about 75%,more particularly at least about 80%, more particularly at least about85%, even more particularly at least about 90% homology, mostparticularly at least about 95% homology, and even most particularly atleast about 97% homology, which encode a signal peptide; or allelicvariants and subsequences of SEQ ID NO: 2 which encode signal peptidefragments which have the ability to direct a polypeptide into or acrossa cell membrane, e.g. into a cell's secretory pathway. For purposes ofthe present invention, the degree of homology between two nucleic acidsequences is determined by the Wilbur-Lipman method (Wilbur and Lipman,1983, Proceedings of the National Academy of Science USA 80: 726-730)using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.)with an identity table and the following multiple alignment parameters:Gap penalty of 10 and gap length penalty of 10. Pairwise alignmentparameters are Ktuple=3, gap penalty=3, and windows=20.

In a third aspect, the first nucleotide sequence of the presentinvention encodes a signal peptide, wherein said first nucleotidesequence hybridize under stringency conditions with the nucleotides ofSEQ ID NO: 2, or its complementary strand (J. Sambrook, E. F. Fritsch,and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2dedition, Cold Spring Harbor, N.Y.).

The nucleotide sequence of SEQ ID NO: 2 or a subsequence thereof, aswell as the amino acid sequence of SEQ ID NO: 1 or a fragment thereofmay be used to design a nucleic acid probe to identify and clone DNAencoding signal peptides from strains of different genera or speciesaccording to methods well known in the art. In particular, such probescan be used for hybridization with the genomic or cDNA of the genus orspecies of interest, following standard Southern blotting procedures, inorder to identify and isolate the corresponding gene therein. Suchprobes can be considerably shorter than the entire sequence, but may inparticular be at least 15, such as at least 25, or more particularly atleast 35 nucleotides in length. Both DNA and RNA probes can be used. Theprobes may typically be labelled for detecting the corresponding gene(for example, with 32P, 3H, 35S, biotin, or avidin). Such probes areencompassed by the present invention.

Thus, a genomic DNA or cDNA library prepared from such other organismsmay be screened for DNA which hybridizes with the probes described aboveand which encodes a signal peptide. Genomic or other DNA from such otherorganisms may be separated by agarose or polyacrylamide gelelectrophoresis, or other separation techniques. DNA from the librariesor the separated DNA may be transferred to and immobilized onnitrocellulose or other suitable carrier material. In order to identifya clone or DNA which is homologous with SEQ ID NO: 2 or a subsequencethereof, the carrier material is used in a Southern blot. For purposesof the present invention, hybridization indicates that the nucleic acidsequence hybridizes to a labelled nucleic acid probe corresponding tothe nucleic acid sequence shown in SEQ ID NO: 2, its complementarystrand, or a subsequence thereof, under stringency conditions definedherein. Molecules to which the nucleic acid probe hybridizes under theseconditions can be detected using X-ray film.

In a particular aspect, the nucleic acid probe is a nucleotide sequencewhich encodes the signal peptide of SEQ ID NO: 1, or a subsequencethereof. In another particular aspect, the nucleic acid probe is SEQ IDNO: 2. In another particular aspect, the nucleic acid probe is thesignal peptide sequence of the cutinase gene contained in Humicolainsolens DSM 1800 (SEQ ID NO: 2 of WO 96/13580).

For short probes which are about 15 nucleotides to about 60 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at 5° C. to 10° C. belowthe calculated Tm using the calculation according to Bolton and McCarthy(1962, Proceedings of the National Academy of Sciences USA 48: 1390) in0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1×Denhardt'ssolution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate,0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southernblotting procedures.

For short probes which are about 15 nucleotides to about 60 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated Tm.

In a fourth aspect, the first nucleotide sequence may encode variants ofthe signal peptide having an amino acid sequence of SEQ ID NO: 1comprising a substitution, deletion, and/or insertion of one or moreamino acids.

The amino acid sequences of the variant signal peptides may differ fromthe amino acid sequence of SEQ ID NO: 1 by an insertion or deletion ofone or more amino acid residues and/or the substitution of one or moreamino acid residues by different amino acid residues.

Particularly, amino acid changes may be of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe activity of the signal peptide; or small deletions, typically of oneto about 5 amino acids.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter the specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly as well as these inreverse.

Polypeptide

The second nucleotide sequence of the preset invention encodes apolypeptide encoded of the present invention. Said polypeptide may benative or heterologous to the fungal host cell in which it is produced.

The term “polypeptide” is not meant herein to refer to a specific lengthof the encoded product and, therefore, encompasses peptides,oligopeptides, and proteins. The term “heterologous polypeptide” isdefined herein as a polypeptide which is not native to the fungal cell,a native polypeptide in which modifications have been made to alter thenative sequence, or a native polypeptide whose expression isquantitatively altered as a result of a manipulation of the geneencoding the polypeptide by recombinant DNA techniques. The fungal cellmay contain one or more copies of the nucleotide sequence encoding thepolypeptide.

In particular, the polypeptide may be a hormone or hormone variant, anenzyme, a receptor or portion thereof, an antibody or portion thereof,an allergen or a reporter. In a particular aspect, the polypeptide maybe an allergen originating from Dermatophagoides pteronyssinus,Dermatophagoides farinae, Dermatophagoides siboney, Dermatophagoidesmicroceaus, Blomia tropicalis and Euroglyphus maynei, or an allergenfrom one of said organisms which subsequently have been modified. Moreparticularly, said allergen may be Der p 1, e.g. Der p 1 fromDermantophagoides pteronyssinus (SEQ ID NO: 3). In particular thepolypeptide may be the sequence of amino acids 19-320 of SEQ ID NO: 3.In a more particular embodiment the polypeptide may be a variant of thepolypeptide sequence of amino acids 19-320 of SEQ ID NO: 3. Moreparticularly said variant may be a S54X or N52X, wherein “X” denotes anyamino acid, as said disrupt the N-glycosylation site of Der p 1, inparticular said variants may be S54N or N52Q. However, other variants ofDer p 1 are envisioned.

In another particular embodiment the polypeptide may be an enzyme, suchas an oxidoreductase, transferase, hydrolase, lyase, isomerase, orligase. In a more particular aspect, the polypeptide may be anaminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase,cellulase, cellobiohydrolase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, endoglucanase, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phospholipase, phytase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transglutaminase, xylanase, or beta-xylosidase. An exampleof a relevant cellulase includes but is not limited to a cellulase fromMucor cicinellides, e.g. M. cicinellides IFO4554 (SEQ ID NO: 4).Examples of relevant proteolytic enzymes include but are not limited tocystein proteases, e.g. cystein protease 5 from Trifolium repens L (SEQID NO: 5), especially amino acids 109-327 of SEQ ID NO: 5 which encodethe mature peptide, or trypsin, e.g. trypsin from Fusarium oxysporium(SEQ ID NO: 7). An example of a relevant phytase includes but is notlimited to a phytase from Peniophora lycii, e.g. P. lycii CBS 686.96(SEQ ID NO: 9).

The second nucleotide sequence encoding a polypeptide of the presentinvention may be obtained from any prokaryotic, eukaryotic, or othersource. For purposes of the present invention, the term “obtained from”as used herein in connection with a given source shall mean that thepolypeptide is produced by the source or by a cell in which a gene fromthe source has been inserted.

The techniques used to isolate or clone a nucleotide sequence encoding apolypeptide of interest are known in the art and include isolation fromgenomic DNA, preparation from cDNA, or a combination thereof. Thecloning of the second nucleotide sequence from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR).See, for example, Innis et al., 1990, PCR Protocols: A Guide to Methodsand Application, Academic Press, New York. The cloning procedures mayinvolve excision and isolation of a desired nucleotide fragmentcomprising the nucleotide sequence encoding the polypeptide, insertionof the fragment into a vector molecule, and incorporation of therecombinant vector into the mutant fungal cell where multiple copies orclones of the nucleotide sequence will be replicated. The secondnucleotide sequence may be of genomic, cDNA, RNA, semisynthetic,synthetic origin, or any combinations thereof.

In the methods of the present invention, the polypeptide may also be afused or hybrid polypeptide in which another polypeptide is fused at theN-terminus or the C-terminus of the polypeptide or fragment thereof. Afused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding one polypeptide to the second nucleotidesequence (or a portion thereof) encoding the polypeptide of the presentinvention. Techniques for producing fusion polypeptides are known in theart, and include, ligating the coding sequences encoding thepolypeptides so that they are in frame and expression of the fusedpolypeptide is under control of the same promoter(s) and terminator. Thehybrid polypeptide may comprise a combination of partial or completepolypeptide sequences obtained from at least two different polypeptideswherein one or more may be heterologous to the mutant fungal cell.

Nucleic Acid Construct

The present invention also relates to a nucleic acid constructcomprising a first nucleotide sequence signal peptide of the presentinvention operably linked to a second nucleotide sequence encoding apolypeptide of the present invention.

“Nucleic acid construct” is defined herein as a nucleotide molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacids combined and juxtaposed in a manner that would not otherwise existin nature. The term nucleic acid construct is synonymous with the termexpression cassette when the nucleic acid construct contains a codingsequence and all the control sequences required for expression of thecoding sequence.

A second nucleotide sequence encoding a polypeptide may be furthermanipulated in a variety of ways to provide for expression of thepolypeptide. Manipulation of the nucleotide sequence prior to itsinsertion into a vector may be desirable or necessary depending on theexpression vector. The techniques for modifying nucleotide sequencesutilizing recombinant DNA methods are well known in the art.

In the methods of the present invention, the nucleic acid construct maycomprise one or more native control sequences or one or more of thenative control sequences may be replaced with one or more controlsequences foreign to the first and/or second nucleotide sequence of thenucleic acid construct for improving expression of the second nucleotidesequence encoding a polypeptide in a host cell.

The term “control sequences” is defined herein to include all componentswhich are necessary or advantageous for the expression of thepolypeptide encoded by the second nucleotide sequence. Each controlsequence may be native or foreign to the second nucleotide sequenceencoding the polypeptide. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,signal peptide sequence of the present invention, and transcriptionterminator. At a minimum, the control sequences include a signal peptidesequence of the present invention, and transcriptional and translationalstop signals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the second nucleotide sequence encodingthe polypeptide.

The control sequence may be an appropriate promoter sequence, which isrecognized by a host cell for expression of the nucleotide sequence. Thepromoter sequence contains transcriptional control sequences whichmediate the expression of the polypeptide. The promoter may be anysequence which shows transcriptional activity in the host cell of choiceincluding mutant, truncated, and hybrid promoters, and may be obtainedfrom genes encoding extracellular or intracellular polypeptides eitherhomologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase, Fusarium oxysporum trypsin-like protease (WO96/00787), Trichoderma reesei cellobiohydrolase I, Trichoderma reeseicellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichodermareesei endoglucanase II, Trichoderma reesei endoglucanase III,Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V,Trichoderma reesei xylanase I, Trichoderma reesei xylanase II,Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter (ahybrid of the promoters from the genes for Aspergillus niger neutralalpha-amylase and Aspergillus oryzae triose phosphate isomerase); andmutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters include but are not limited to thoseobtained from the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase(ADH1,ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase(TPI), Saccharomyces cerevisiae metallothionine (CUP1), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8: 423-488.

The control sequence may be a suitable transcription terminatorsequence, which is recognized by a host cell to terminate transcription.The terminator sequence is operably linked to the 3′ terminus of thenucleotide sequence encoding the polypeptide. Any terminator which isfunctional in the host cell of choice may be used in the presentinvention.

Examples of suitable terminators for filamentous fungal host cellsinclude but are not limited to those obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,Aspergillus nidulans anthranilate synthase, Aspergillus nigeralpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Examples of suitable terminators for yeast host cells include but arenot limited to those obtained from the genes for Saccharomycescerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), andSaccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Otheruseful terminators for yeast host cells are described by Romanos et al.,1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence may generally be operably linked tothe 5′ end of the nucleotide sequence encoding a polypeptide. Any leadersequence that is functional in the host cell of choice may be used inthe present invention.

Examples of suitable leaders for filamentous fungal host cells includebut are not limited to those obtained from the genes for Aspergillusoryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

Suitable leaders for yeast host cells include but are not limited tothose obtained from the genes for Saccharomyces cerevisiae enolase(ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase,Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase(ADH2/GAP).

The control sequence may also be a polyadenylation sequence, which isoperably linked to the 3′ end of the nucleotide sequence encoding apolypeptide and which, when transcribed, is recognized by the host cellas a signal to add polyadenosine residues to transcribed mRNA. Anypolyadenylation sequence which is functional in the host cell of choicemay be used in the present invention.

Examples of suitable polyadenylation sequences for filamentous fungalhost cells include but are not limited to those obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,Aspergillus nidulans anthranilate synthase, Fusarium oxysporumtrypsin-like protease, and Aspergillus niger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells include but arenot limited to those described by Guo and Sherman, 1995, MolecularCellular Biology 15: 5983-5990.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. Examples of propeptide coding region include but are notlimited to those obtained from the genes for Dermantophagoidespteronyssinus Der p 1, or other genes obtained from Dermantophagoides,Fusarium oxysporum trypsin, Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion. If a propeptide is present a cleavable site may in oneembodiment be present between the propeptide and the mature polypeptide.The term “cleavable site” is to be understood as an amino acid sequencewhich is recognized by a proteolytic enzyme capable of cleaving thepolypeptide at this site. Examples of such site include a kex-site, inparticular a kex-II site.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the TAKA alpha-amylase promoter, Aspergillus nigerglucoamylase promoter, and Aspergillus oryzae glucoamylase promoter maybe used as regulatory sequences. Other examples of regulatory sequencesare those which allow for gene amplification. In eukaryotic systems,these include the dihydrofolate reductase gene which is amplified in thepresence of methotrexate, and the metallothionein genes which areamplified with heavy metals. In these cases, the nucleotide sequenceencoding the polypeptide would be operably linked with the regulatorysequence.

Expression Vector

The present invention also relates to a recombinant expression vectorcomprising a nucleic acid construct of the present invention. Besidescomprising a first and a second nucleotide sequence encoding a signalpeptide and a polypeptide, respectively of the present invention saidexpression vector may in particular comprise a transcriptional andtranslational stop signal. The various nucleotide and control sequencesdescribed above may be joined together to produce a recombinantexpression vector which may include one or more convenient restrictionsites to allow for insertion or substitution of the promoter and/ornucleotide sequence encoding the polypeptide at such sites.Alternatively, the nucleic acid construct may be inserted into anappropriate vector for expression for expression of the polypeptideencoded by the second nucleotide sequence. In creating the expressionvector, the second nucleotide sequence encoding the polypeptide of thepresent invention is located in the vector so that said sequence isoperably linked with a signal peptide sequence of the present inventionand one or more appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the nucleotide sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids.

The vectors of the present invention may in particular contain one ormore selectable markers which permit easy selection of transformedcells. A selectable marker is a gene the product of which provides forbiocide or viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Suitable markers for yeast host cells include,but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), as well as equivalents thereof. Inparticular for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention may in particular contain anelement(s) that permits stable integration of the vector into the hostcell's genome or autonomous replication of the vector in the cellindependent of the genome.

For integration into the host cell genome, the vector may rely on thesecond nucleotide sequence encoding the polypeptide or any other elementof the vector for stable integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleotide sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleotide sequences may enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements should in particular contain a sufficient numberof nucleotides, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleotide sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication which functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6. The origin of replicationmay be one having a mutation which makes its ability to functiontemperature-sensitive in the host cell (see, e.g., Ehrlich, 1978,Proceedings of the National Academy of Sciences USA 75: 1433).

Examples of origins of replication useful in a filamentous fungal hostcell are AMA1 and ANSI (Gems et al., 1991, Gene 98:61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a nucleotide sequence encoding a polypeptide maybe inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleotide sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleotide sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleotide sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention relates to methods in which polypeptides areproduced in a fungal host cell and to a recombinant host cellscomprising a nucleic acid construct of the present invention.

A vector comprising a first nucleotide sequence encoding a signalpeptide of the present invention operably linked to a second nucleotidesequence encoding a polypeptide is introduced into a fungal host cell sothat the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be any fungal cell useful in the methods of thepresent invention.

“Fungi” as used herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra).

In a particular aspect, the fungal host cell is a yeast cell. “Yeast” asused herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In a more particular aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most particular aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, e.g., S. cerevisiae YNG318,Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyceskluyveri, Saccharomyces norbensis, Saccharomyces oviformis,Kluyveromyces lactis or Yarrowia lipolytica cell.

In another particular aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are characterized by a mycelial wallcomposed of chitin, cellulose, glucan, chitosan, mannan, and othercomplex polysaccharides. Vegetative growth is by hyphal elongation andcarbon catabolism is obligately aerobic. In contrast, vegetative growthby yeasts such as Saccharomyces cerevisiae is by budding of aunicellular thallus and carbon catabolism may be fermentative.

In a more particular aspect, the filamentous fungal host cell is a cellof a species of, but not limited to, Acremonium, Aspergillus, Fusarium,Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia,Tolypocladium, or Trichoderma. In an even more particular aspect, thefilamentous fungal host cell is an Aspergillus awamori, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus nigeror Aspergillus oryzae cell. In another even more particular aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another even more particular aspect, the filamentousfungal host cell is a Humicola insolens, Humicola lanuginosa, Mucormiehei, Myceliophthora thermophila, Neurospora crassa, Penicilliumpurpurogenum, Thielavia terrestris, Trichoderma harzianum, Trichodermakoningii, Trichoderma longibrachiatum, Trichoderma reesei, orTrichoderma viride cell.

In a most particular aspect, the Fusarium venenatum cell is Fusariumvenenatum A3/5, which was originally deposited as Fusarium graminearumATCC 20334 and recently reclassified as Fusarium venenatum by Yoder andChristianson, 1998, Fungal Genetics and Biology 23: 62-80 and O'Donnellet al., 1998, Fungal Genetics and Biology 23: 57-67; as well astaxonomic equivalents of Fusarium venenatum regardless of the speciesname by which they are currently known. In another particular aspect,the Fusarium venenatum cell is a morphological mutant of Fusariumvenenatum A3/5 or Fusarium venenatum ATCC 20334, as disclosed in WO97/26330.

In another most particular aspect, the Trichoderma cell is Trichodermareesei ATCC 56765, Trichoderma reesei ATCC 13631, Trichoderma reesei CBS526.94, Trichoderma reesei CBS 529.94, Trichoderma longibrachiatum CBS528.94, Trichoderma longibrachiatum ATCC 2106, Trichodermalongibrachiatum CBS 592.94, Trichoderma viride NRRL 3652, Trichodermaviride CBS 517.94, or Trichoderma viride NI BH FERM/BP 447.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474. Suitable procedures for transformation of Trichodermareesei host cells are described in Penttila et al., 1987, Gene 61:155-164, and Gruber et al., 1990, Curr Genet. 18(1):71-6. Suitablemethods for transforming Fusarium species are described by Malardier etal., 1989, Gene 78: 147-156 and WO 96/00787. Yeast may be transformedusing the procedures described by Becker and Guarente, In Abelson, J. N.and Simon, M. I., editors, Guide to Yeast Genetics and MolecularBiology, Methods in Enzymology 194: 182-187, Academic Press, Inc., NewYork; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen etal., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.

Materials and Methods Strains and Plasmids Strains

E. coli DH12S (available from Gibco BRL) is used for yeast plasmidrescue.

Saccharomyces cerevisiae YNG318: MATa Dpep4[cir+] ura3-52, leu2-D2, his4-539 is described in J. Biol. Chem. 272 (15): 9720-9727, 1997).

Plasmids

All yeast expression vectors are S. cerevisiae and E. coli shuttlevectors under the control of TPI promoter, constructed from pJC039described in WO 00/10038.

Genes

Der p 1 from Dermatophagoides pteronyssinus: NCBI accession number:P08176, the amino acid sequence is shown in SEQ ID NO: 3.

The Cystein protease gene from Trifolium repens L (white clover) is thesequence which is deposited as EMBL: AY192363.

The alpha-factor (pheromone required for yeast mating) gene is thesequence in the commercial available pPICZα A vector from Invitrogen.

The trypsin gene from Fusarium oxysporum is the sequence which isdeposited as EMBL: S63827 and for which the cDNA sequence is shown inSEQ ID NO: 6.

The phytase gene from Peniophora lycii strain CBS 686.96 is the sequencewhich is deposited as EMBL: PLY310696 and for which the cDNA sequence isshown in SEQ ID NO: 8.

Primers For expression of a cellulase from Mucor circinellides For cDNA cloning: MCE-BC1 F (44 mer): (SEQ ID NO: 10)5′-CAACTGGTGATCACCACCATGAAGTTCACCGTTGCTATTACTT C-3′ MCE-Nru R (39 mer):(SEQ ID NO: 11) 5′-TCTCGAGCTCGCGATTACTTTCTTTCGCAACCTGAGCGAG-3′For yeast vector construction: M61 F (50 mer): (SEQ ID NO: 12)5′-CCAGCTTCCGCAAACAAAGTCGCCAACATGAAGTTCACCGTTGCTA TTAC-3′M61Cutisig F (50 mer): (SEQ ID NO: 13)5′-CGCCAGCCTTGTTGCTGCTCTCCCCGCCGCTTCTTGCAGCTCTGTC TATG-3′C-term R61 R (49 mer): (SEQ ID NO: 14)5′-TAATTACATGATGCGGCCCTCTAGATTACTTTCTTTCGCAACCTGA GCG-3′For expression of cystein protease from Trifolium  reDens Lalpha-signal-Muni (49 mer): (SEQ ID NO: 15)5′-ATAAACGACGGGACCCGGGGATCCAATTGATGAGATTCCCATCAAT TTT-3′alpha-signal-CysPro R (42 mer): (SEQ ID NO: 16)5′-GCCCACGATGGAGAAATCGCGAGCTTCAGCTTCTCTTTTCTC-3′alpha-signal-CysPro F(42 mer): (SEQ ID NO: 17)5′-GAAAAAAGAGAAGCTGAAGCTCGCGATTTCTCCATCGTGGGC-3′ Spe-CysPro R (50 mer):(SEQ ID NO: 18) 5′-ACTAATTACATGATGCGGCCCACTAGTTCATTTCTTCTTAGTAGGATAAG-3′ Cuti-Sig-CysPro (50 mer): (SEQ ID NO: 19)5′-GCACCGCCAGCCTTGTTGCTGCTCTCCCCCGCGATTTCTCCATCGT GGGC-3′CysPro C-term (50 mer): (SEQ ID NO: 20)5′-TAATTACATGATGCGGCCCGCGGCCGCTCATTTCTTCTTAGTAGGA TAAG-3′For expression of trypsin from Fusarium yeast-F (43 mer):(SEQ ID NO: 21) 5′-ACGACGGTACCCGGGGATCAAGCTTATGGTCAAGTTCGCTTCC-3′yeast-R (43 mer): (SEQ ID NO: 22)5′-AACTAATTACATGATGCGGCCCTCTAGATTAAGCATAGGTGTC-3′ cuti-pre (45 mer):(SEQ ID NO: 23) 5′-CGTTCCTGAACTTGTTGCCCGGGTTGGTGGCACTTCTGCCAGCG C-3′TP2-Kex F (32 mer): (SEQ ID NO: 24)5′-GTTCCTGAACTTGTTCGGCGGGTTGGTGGCAC-3′ TP2-Kex R (32 mer):(SEQ ID NO: 25) 5′-GTGCCACCAACCCGCCGAACAAGTTCAGGAAC-3′Cuti-pre F (29 mer): (SEQ ID NO: 26) 5′-GTTGCTGCTCTCCCCGTTGGTGGCACTTC-3′Cuti-pre R (29 mer): (SEQ ID NO: 27) 5′-GAAGTGCCACCAACGGGGAGAGCAGCAAC-3′CUTIpre-TPpro F (42 mer): (SEQ ID NO: 28)5′-TCCTCAGGAGATCCCCAACATTGTTGGTGGCACTTCTGCCAG-3′CUTIpre-TPpro R (40 mer): (SEQ ID NO: 29)5′-GTTGGGGATCTCCTGAGGAGCGGGGAGAGCAGCAACAAGG-3′For expression of a phytase from Peniophora PP 1F (50 mer):(SEQ ID NO: 30) 5′-AAACGACGGTACCCGGGGATCAAGCTTATGGTTTCTTCGGCATTCGCACC-3′ PP R (50 mer): (SEQ ID NO: 31)5′-ACTAATTACATGATGCGGCCCTCTAGACTATTCCGACGGAACAAAG CCGC-3′PP 2F (50 mer): (SEQ ID NO: 32)5′-CCGCCAGCCTTGTTGCTGCTCTCCCCCAGCTACCTATCCCCGCACA AAAC-3′

Medium and Substrates

RS-25: 40 g/L soy bean powder, 40 g/L glucose, 10 g/L KH₂PO₄, 0.25 g/LMgSO₄, 0.01 g/L FeSO₄, 2.5 g/L NH₄NO₃; pH 6

YPD: 20 g/L Glucose, 20 g/L Pepton and 10 g/L Yeast extract

10× Basal solution: 66.8 g/l Yeast nitrogen base w/o amino acids(DIFCO), 100 g/l succinate and 60 g/l NaOH.

SC-glucose (or SC-medium): 100 ml/l 20% glucose (i.e., a finalconcentration of 2%=2 g/100 ml), 4 ml/l 5% threonine, 10 ml/l 1%tryptophan, 25 ml/l 20% casamino acids and 100 ml/l 10× basal solution.This solution was sterilized using a filter of a pore size of 0.20micron. Agar and H₂O (approx. 761 ml) is autoclaved together, and theseparately sterilized SC-glucose solution is added to the agar solution.

PEG/LiAc solution: 50 ml 40% PEG4000 (sterilized by autoclaving) and 1ml 5 M lithium acetate (sterilized by autoclaving).

Methods Yeast Transformation

This method was used in examples 2-5.

To transform yeast the in vivo recombinant mechanism was utilized bywhich it is possible for yeast to recombine a vector sequence and PCRfragments in vivo to create an expression vector, if both the vectorsequence and the PCR fragments have the same flanking regions.

A DNA mixture was prepared by mixing 0.5 Microl of vector (EcoRI-NotIdigested) and 1 Microl of PCR fragments. S. cerevisiae YNG318 competentcells were thawed on ice. One hundred Microl of the cells were mixedwith the DNA mixture and 10 Microl of carrier DNA (Clontech) in 12 mlpolypropylene tubes (Falcon 2059). To this 0.6 ml PEG/LiAc solution wasadded and mixed gently and then incubated for 30 min at 30° C., and 200rpm. Thereafter it was incubated for 30 min at 42° C. (heat shock)before transferring it to an eppendorf tube and centrifugation for 5sec. The supernatant was removed and resolved in 3 ml of YPD. The cellsuspension was then incubated for 45 min at 200 rpm at 30° C. before itwas poured on to SC-glucose plates.

PCR Reaction

Unless otherwise indicated the PCR reactions were carried out under thefollowing conditions:

The PCR reaction contained 38.9 MicroL H2O, 5 MicroL 10× reactionbuffer, 1 MicroL Klen Taq LA (Clontech), 4 MicroL 10 mM dNTPs, 0.3MicroL×2 100 pmol/MicroL primer and 0.5 MicroL template DNA and wascarried out under the following conditions: 30 cycles of 10 sec at 98°C. and 90 sec at 68° C., and a final 10 min at 68° C.

Sandwich ELISA

Immunoplates (Nunc Maxisorb; Nunc-Nalgene) were coated overnight at 4°C. with at suitable dose of polyclonal rabbit anti Der p 1 antibody. Theplates were then washed thoroughly with 0.15 M Phosphate Buffered Saline(PBS) containing 0.05% Tween 20 (PBST), and remaining binding sites areblocked with PBS with 2% skim milk powder, 1 h at room temperature.Samples, it can be purified, semi-purified recombinant group 1 mitepolypeptide variant allergen or crude culture broth containing proteinof interest, were added in a suitable dose or dose-range. The plateswere then washed thoroughly with 0.15 M PBST before the allergens weredetected by incubation with biotinylated monoclonal anti Der p 1antibody (INDOOR) 1 h at room temperature. The plates were then washedagain in 0.15 M PBST before conjugated with complexes ofStreptavidin:Horse Radish Peroxidase (Kierkegaard & Perry) for 1 h atroom temperature. The washing step was repeated and then the plates weredeveloped by adding 3,3′,5,5′-tetramethylbenzidine hydrogen peroxide(TMB Plus, Kem-En-Tec) before the reaction was stopped by addition of0.2 M H2SO4. The optical density (OD) at 450 nm reflected allergenbinding to the immunoglobin, and it was then possible to detect and alsodetermine the amount of allergen bound by comparing with the dataobtained for natural Der p 1 (available from Indoor biotechnologies,NA-DP1) which was included in the ELISA in a known concentration doserange.

Other Methods

E. coli transformation to rescue yeast plasmid was carried out byelectroporation (BIO-RAD Gene Pulser).

DNA Plasmids were prepared with the Qiagen® Plasmid Kit. DNA fragmentsand recovered from agarose gel by the Qiagen gel extraction Kit.

PCR was carried out by the PTC-200 DNA Engine.

The ABI PRISM™ 310 Genetic Analyzer was used for determination of allDNA sequences. Yeast total DNA was extracted by the Robzyk and Kassir'smethod described in Nucleic Acids Research 20(14): 3790 (1992).

Enzyme Assays Cystein Protease Assay

96 well microtiter plate assay:

The following was added to each well: 10 MicroL 0.5 M Sodium acetatebuffer (pH 5), 10 MicroL 2 M NaCl (comprising 700 MicroLmercaptethano/100 ml), 45 MicroL D.W. (distilled water) and 10 MicroLenzyme sample. The plate was then incubated at room temperature for 5-10min (for maturation of the peptidase) before 25 MicroL 40 MicroMZ-Phe-Arg-MCA (0.1% DMSO) was added. Finally, the emission of MCA(4-methyl-coumaryl-7-amide) at 460 nm was measured in a fluorometer.

Trypsin Assay (PNA Assay for Fusarium Trypsin) Substrate:

Fifty mg of N alpha-Benzoyl-DL-Arginine-p-Nitroanilide (BAPNA, SigmaB-4875) was dissolved in 1 ml DMSO and kept at −20° C. This solution wasdiluted 100× in the assay buffer just before use.

Assay Buffer:

50 mM Borate-NaOH (pH 10.5)+2 mM CaCl₂

Method:

20 Microl sample and 200 Microl assay buffer were mixed in a 96-wellmicrotiter tray and Delta OD/min was measured at 405 nm for 5 min.

Phytase Assay

Ten MicroL diluted enzyme samples (diluted in 0.1 M sodium acetate,0.01% Tween20, pH 5.5) were added into 250 MicroL of 5 mM sodium phytate(Sigma) in 0.1 M sodium acetate, 0.01% Tween20, pH 5.5 (pH adjustedafter dissolving the sodium phytate; the substrate was preheated) andincubated for 30 minutes at 37° C. The reaction was stopped by adding250 MicroL 10% TCA and free phosphate was measured by adding 500 MicroL7.3 g FeSO₄ in 100 ml molybdate reagent (2.5 g (NH₄)₆Mo70₂₄.4H₂0 in 8 mlH₂SO₄ diluted to 250 ml). The absorbance at 750 nm was measured on 200MicroL samples in 96 well microtiter plates. Substrate and enzyme blankswere included. A phosphate standard curve was also included (0-2 mMphosphate). 1 U equals the amount of enzyme that releases 1 Micromolphosphate/min at the given conditions.

EXAMPLES Example 1 Expression of Der p 1 in S. cerevisiae

The Der p 1 cystein protease from Dermantophagoides pteronyssinus (theamino acid sequence of which is depicted in SEQ ID NO: 3) encoding genewas located in vector pStep 212, which is derived from yeast expressionvector pYES 2.0 (Invitrogen, Kofod et al., 1994, J. Biol. Chem. 269:29182-29189 and Christgau et al., 1994, Biochem. Mol. Biol. Int. 33:917-925).

This plasmid replicated both in E. coli and in S. cerevisiae. In S.cerevisiae Der p 1 was expressed from this plasmid.

Recombinant Der p 1 was expressed with the Der p 1 propeptide and hadthe mutation S54N or N52Q which disrupts the only N-glycosylation motifwithin the mature sequence.

For secretion in yeast two different signal peptides were tested fortheir expression efficiency by introducing them upstream of the encodingpro-Der p 1 gene. Expression constructs with signal peptides were madeby cloning of DNA fragments (Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor, 1989). One signalpeptide was the natural occurring Dermantophagoides pteronyssinus Der p1 signal peptide with the amino acid sequence MKIVLAIASLLALSAVYA (SEQ IDNO: 3) and the other signal peptide was derived from a cutinase fromHumicula insolens with the amino acid sequence MKFFTTILSTASLVAALP (SEQID NO: 1). The yeast strain and the construct with the dust mite Der p 1signal peptide was named pre-pro-Der p 1 and the yeast strain andconstruct with the cutinase signal peptide was named cuti-pro-Der p 1.

The constructs were transformed into S. cerevisiae. For screening ofyeast transformants expressing Der p 1, the transformation solution wasplated on SC-agar plates for colony formation at 30° C. for 3 days.Colonies were inoculated in 50 ml sterile plastic tubes, each tubecontaining 10 mL SC medium. The tubes were fermented in 500 ml baffledErlenmeyer flasks containing 100 ml SC medium at 30° C., 250 rpm for 4days. Culture broth from these fermentations were used for sandwichELISA experiments to determine the concentration of expressed protein.

The expression level of Der p 1 by the pre-pro Der p1 S54N and thepre-pro Der p1 N52Q transformants were determined by sandwich ELISA asdescribed above in the Method section to be at the same level plus/minus8%. It was concluded that the expression level of the two strains withthe pre-pro-Der p 1 was independent of which of the two mutations S54Nand N52Q were used.

In another experiment, 4.5 L culture broth of pre-pro-Der p 1 (N52Q) andcuti-pro-Der p 1 (S54N) were fermented for 4 days and samples were takeneach day during fermentation and the amount of Der p 1 was measured bythe Sandwich ELISA as described above to follow the expression level.The ratio between expression yields of cuti-pro-Der p 1 and pre-pro-Derp 1 is shown below.

Fermentation day Expression yield (cuti-pro Der p 1/pre-pro-Der p 1) Day1 8 Day 2 10 Day 3 8 Day 4 6 For each day of fermentation thecuti-pro-Der p 1 expressed between 6-10 times more Der p 1 thanpre-pro-Der p 1 which showed that the cutinase signal peptide in frontof pro-Der 1 increased the expression level of pro-Der p 1 proteincompared to the use of natural dust mite Der p 1 signal peptide.

Example 2 Expression of a Cellulase from Mucor circinellides in S.Cerevisiae

Mucor circinellides IFO4554 was cultivated in shake flask containingRS-25+0.5% lactose medium for one day at 30° C. The mycelium wascollected and used for mRNA preparation, which was subsequently reversetranscribed into cDNA. An approximately 1 kb fragment was amplified byPCR from the cDNA by using the MCE-BC1 F and MCE-Nru R primer pair andthe following PCR conditions: 2 min at 94° C.; 35 cycles of 1 min at 94°C., 1 min at 45° C. and 2.5 min at 72° C.; and finally 8 min at 72° C.The amplified fragment was cloned into T-vector (Novagene).

The fragment (the Mucor cellulase gene) in T-vector was re-amplifiedwith the primer pairs, M61 F and C-term R61R, and M61Cutisig F andC-term R61R, where the former pair was used for the construct with theoriginal signal sequence (the signal sequence of the Mucor cellulasegene) and the latter pair was used to construct a nucleic acid sequencecomprising the signal sequence from the H. insolens cutinase (SEQ ID NO:2) and the Mucor cellulase gene.

Yeast was transformed by introducing the resulting PCR fragments into S.cerevisiae YNG318 together with the pJC039 vector digested with HindIIIand XbaI, and PvuII and XbaI, respectively.

The obtained transformants were cultivated in 24 well plates containingYPD medium at 30° C. and 180 rpm for 3 days. The culture supernatantswere applied to holes in an agar plated containing 0.2% CMC (carboxymethyl cellulose), pH 8.5. In below table the size of the halossurrounding holes containing culture supernatant from yeast cellsexpressing the cellulase from Mucor with either the H. insolens cutinasesignal peptide or with the signal peptide from the Mucor cellulase gene.As the cellulase is capable of degrading the CMC thereby creating thehalo the size of the halo correlates to the amount of cellulase presentin the culture supernatant which was placed in the particular hole. Thusa large halo indicates high amounts of cellulase while a small haloindicates a low amount of cellulase.

Halo size in CMC plate Mucor cellulase with H. insolens cutinase +++++signal peptide Mucor cellulase with its own signal peptide (+) Theresults indicate that the expression level in yeast of Mucor cellulaseis much higher when it is expressed with the signal peptide sequencefrom the H. insolens cutinase gene than when expressed with its ownsignal peptide sequence (Mucor cellulase gene).

Example 3 Expression of Cystein Protease from Trifolium repens L in S.cerevisiae

To construct an expression vector comprising the signal peptide fromalpha-factor and the cystein protease from Trifolium repens L, the DNAfragment encoding the alpha-factor signal peptide was re-amplified witha primer pair, alpha-signal-MunI and alpha-signal-CysProR. The geneencoding pro-mature region of the cystein protease from Trifolium repensL was re-amplified with a primer pair, alpha-signal-CysPro F and Spe-CysProR using the EMBL:AY192363 sequence as template. Yeast was transformedby mixing these two PCR fragments together with pJC039 vector digestedwith Hind III and Xba I and introduced into S. cerevisiae YNG318.

To construct an expression vector comprising the signal peptide from theH. insolens cutinase and the cystein protease from Trifolium repens L,the gene encoding the pro-mature region of the cystein protease fromTrifolium repens L was re-amplified with a primer pair, Cuti-Sig-CysProand CysPro C-term. Yeast was transformed by mixing the obtained PCRfragment with pJC039 vector digested with Hind III and Xba I andintroduced into S. cerevisiae YNG318.

The obtained transformants were cultivated in 24 well plate containing 1ml of YPD at 27° C. for 3 days and then the supernatants were tested forcystein protease activity as described in the Methods section. Themeasurements of the cystein protease activity are shown in below table.

Cystein protease activity (fluorescence emission/min) Cystein proteasewith H. insolens cutinase 4.446 × 10⁶ signal peptide Cystein proteasewith alpha-factor signal 4.073 × 10⁵ peptide The results indicate thatthe expression level in yeast of the cystein protease from white cloveris 10 times higher when the protease is expressed with the H. insolenscutinase signal peptide than with the alpha-factor signal peptide.

Example 4 Expression of Trypsin from Fusarium in S. cerevisiae

The trypsin gene from Fusarium was re-amplified with the primer pairs,yeast-F and yeast-R, and cuti-pre and yeast-R using the EMBL: S63827sequence as template, i.e. the cDNA sequence shown in SEQ ID NO: 6. Theyeast-F/yeast-R primer pair was used to construct a sequence comprisinga nucleic acid sequence encoding the signal peptide, the pro-region andthe mature part of the trypsin gene, while the cuti-pre/yeast-R primerpair was used to construct a nucleic acid sequence comprising the signalpeptide and pro-region from the H. insolens cutinase and the maturetrypsin gene from Fusarium. Yeast was transformed introducing theresulting PCR fragments into S. cerevisiae YNG318 together with thepJC039 vector digested with HindIII and XbaI, and PvuII and XbaI.

The obtained transformants (pTM-TP1 and pTM-TP2) were cultivated in 24well plates containing YPD medium at 30° C., 180 rpm for 3 days.

pTM-TP1: Fusarium Trypsin signal+Fusarium Trypsin Pro+mature trypsinpTM-TP2: H. insolens cutinase signal+H. insolens cutinase Pro+maturetrypsin

The trypsin activity was measured as described above in the Methodsection. No activity was observed in both culture supernatant even withNeutrase™ (as a maturase) addition. However, the pro-form of trypsin(cutinase pro+mature trypsin) was detected by Western blotting withpTM-TP2.

Therefore, the following constructs were made:

pTM-TP2-Kex: H. insolens cutinase signal peptide+H. insolens cutinasepro-region+KexII site

+mature Fusarium trypsin

pTM-TP2w/o pro: H. insolens cutinase signal peptide+mature FusariumtrypsinpTM-TP2 TPpro: H. insolens cutinase signal peptide+Fusarium trypsinpro-region+Fusarium mature trypsin

Each of the primer pairs, TP2-Kex F and R, Cuti-pre F and R andCUTI-pre-TPpro F and R, were used to prepare pTM-TP2-Kex, pTM-TP2w/o proand pTM-TP2 TPpro, respectively by mixing them with pTM-TP2 vectordigested with EagI and introducing this mixture into S. cerevisiaeYNG318.

The obtained transformants were cultivated in 24 well plates containingYPD medium at 30° C., 180 rpm for 3 days.

Activity w/o Activity Western blot Western blot Signal Neutrase with ofcell extract of supernatant Construct peptide Pro-region (as a maturase)Neutrase (intracellular) (extracellular) pTM-TP1 Trypsin Trypsin − −Pro-form − pTM-TP1Kex Trypsin Trypsin − − Pro-form − (+kex site) pTM-TP2Cutinase Cutinase − − − Pro-form pTM-TP2Kex Cutinase Cutinase − − N.T.N.T. (+kex site) pTM-TP2 w/o pro Cutinase None − − − Larger than maturepTM-TP2TPpro Cutinase Trypsin − + − Pro-form wherein “−” means that noactivity or no band in the Western blot could be detected, “+” meansthat trypsin activity was detectable, “pro-form” means that it was thepro-form of the trypsin which was detectable, “N.T.” means “not tested”and for the pTM-TP2 w/o pro means that a protein larger than the maturewas detectable, however this construct was designed not to include apro-region, thus it is not a pro-form. Trypsin activity was detectedwith pTM-TP2TPpro vector when Neutrase ™ was added to the supernatant(final conc. 0.5 AU/ml). The results indicate that the Fusariumtrypsin's own signal peptide does not work efficiently in yeast forexpression of trypsin, while the H. insolens cutinase signal peptidedoes. The pro-region (also called the pro-sequence) from the Fusariumtrypsin (7 amino acids) seems to be necessary for proper folding andactivation of the trypsin.

Example 5 Expression of a Phytase from Peniophora in S. cerevisiae

The Peniophora phytase gene was amplified with the primer pairs (PP1 Fand PP R, PP2 F and PPR using the EMBL: PLY310696 sequence as template,i.e. the cDNA sequence shown in SEQ ID NO: 8. The PP1 F/PP R primer pairwas used to construct a nucleic acid sequence encoding the signalpeptide from the Peniophora phytase gene and the mature protein ofPeniophora phytase, while the PP2 F/PPR primer pari was used toconstruct a nucleic acid sequence encoding the signal peptide from theH. insolens cutinase gene and the mature Peniophora phytase protein.Yeast was transformed by introducing the resulting PCR fragments into S.cerevisiae YNG318 together with the pJCO₃₉ vector digested with HindIIIand XbaI, and PvuII and XbaI.

The obtained transformants were cultivated in 24 well plates and 500ml-shake flasks containing YPD medium at 30° C., 180 rpm for 3 days.

The pTMPP1 construct comprised the original signal peptide sequence fromthe Peniophora phytase, while the pTMPP2 construct comprised the H.insolens cutinase signal peptide sequence. The phytase activity wasmeasured, as described in the Method section, in the culture supernatantof transformants comprising the pTMPP1 or the pTMPP2 construct whichwere culture either in 24-well plates or in a shake flask. The resultsare shown below.

24-well Shake flask pTMPP1 (phytase signal) 1.8 U/ml  1 U/ml pTMPP2(cutinase signal) 6.9 U/ml 13 U/ml The results show that thetransformants comprising the pTMPP2 construct express more phytaseactivity than those comprising the pTMPP1 construct indicating that theH. insolens signal peptide increases expression of the phytase in yeastcompared with the phytase's own signal peptide.

Example 6 Expression of Phytase from Peniophora in Aspergillus oryzae

The constructs comprising the phytase gene with its own signal peptidesequence and the phytase gene with the H. insolens cutinase signalpeptide sequence described in example 5 were used to construct anexpression vector for Aspergillus and transformed into Aspergillus asdescribed in Lassen et al., 2001, Applied and EnvironmentalMicorbiology, 67: 4701-4707. For each of the constructs 49 strains wereisolated, purified and the production of phytase was measured asdescribed in the Method section above by culturing said transformants inshake flasks. When comparing the strains from the two groups ofconstruct which expressed the highest level of phytase, it was foundthat the strain comprising the construct with the cutinase signalpeptide expressed approximately 1.4 times more phytase than the onecomprising the phytase signal peptide. Similarly, when the averageamount of expressed phytase in the 5 strains from each group whichexpressed the highest amount of phytase was compared the strainscomprising the construct with the cutinase signal peptide expressedapproximately 1.3 times more phytase than those comprising the constructwith the phytase signal peptide.

What is claimed is:
 1. A nucleic acid construct comprising a firstnucleotide sequence encoding a signal peptide operably linked to asecond nucleotide sequence encoding an enzyme, wherein the firstnucleotide sequence is foreign to the second nucleotide sequence, the 3′end of the first nucleotide sequence is immediately upstream of thesecond nucleotide sequence, and the first nucleotide sequence isselected from the group consisting of: (a) a nucleotide sequenceencoding a signal peptide amino acid sequence having at least 90%sequence identity with SEQ ID NO: 1, wherein the signal peptide retainsthe ability to direct the enzyme out of a host cell, and (b) anucleotide sequence having at least 90% sequence identity with SEQ IDNO: 2 and that encodes a signal peptide, wherein the signal peptideretains the ability to direct the enzyme out of the host cell.
 2. Thenucleic acid construct of claim 1, wherein the first nucleotide sequenceencodes a signal peptide amino acid sequence having at least 95%sequence identity with SEQ ID NO:
 1. 3. The nucleic acid construct ofclaim 2, wherein the first nucleotide sequence encodes a signal peptideamino acid sequence having at least 97% sequence identity with SEQ IDNO:
 1. 4. The nucleic acid construct of claim 1, wherein the firstnucleotide sequence encodes a signal peptide amino acid sequenceconsisting of SEQ ID NO:
 1. 5. The nucleic acid construct of claim 1,wherein the first nucleotide sequence has at least 95% sequence identitywith SEQ ID NO:
 2. 6. The nucleic acid construct of claim 4, wherein thefirst nucleotide sequence has at least 97% sequence identity with SEQ IDNO:
 2. 7. The nucleic acid construct of claim 1, wherein the firstnucleotide sequence consists of SEQ ID NO:
 2. 8. The nucleic acidconstruct of claim 1, wherein the second nucleotide sequence encodes aheterologous enzyme.
 9. The nucleic acid construct of claim 1, whereinthe enzyme is selected from the group consisting of an oxidoreductase, atransferase, a hydrolase, a lysase, an isomerase, and a ligase.
 10. Thenucleic acid construct of claim 1, wherein the enzyme is selected fromthe group consisting of aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, cellobiohydrolase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,endoglucanase, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase,lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phospholipase, phytase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transglutaminase, xylanase, and beta-xylosidase.
 11. Thenucleic acid construct of claim 1, wherein the enzyme is amylase. 12.The nucleic acid construct of claim 1, wherein the enzyme is phytase.13. A recombinant expression vector comprising the nucleic acidconstruct of claim
 1. 14. A recombinant host cell comprising the nucleicacid construct of claim
 1. 15. The recombinant host cell of claim 14,wherein the fungal host cell is selected from the group consisting ofAcremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora,Neurospora, Penicillium, Thielavia, Tolypocladium, and Trichoderma. 16.The recombinant host cell of claim 14, wherein the fungal host cell isselected from the group consisting of Aspergillus awamori, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, and Aspergillus oryzae.
 17. The recombinant host cell of claim14, wherein the fungal host cell is selected from the group consistingof Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, and Trichoderma viride.
 18. Therecombinant host cell of claim 14, wherein the recombinant host cell isAspergillus and the enzyme is phytase.